Celiac Disease (CD) is a chronic gastrointestinal tract disorder in which ingestion of gluten, present in food products made from wheat, rye, barley and their cross-related varieties, leads to damage of the small intestinal mucosa by an autoimmune mechanism in genetically susceptible individuals (Green P. H. R., Cellier C. “Celiac Disease” N. Engl. J. Med., 2007, 357, 1731-1743; Kagnoff M. F. “Celiac disease: pathogenesis of a model immunogenetic disease” J. Clin. Invest., 2007, 117, 41-9). Mechanisms through which gluten induces its pathogenic effects have been explained in recent years. Both innate and adaptive immunity mechanisms are involved and are responsible for the ultimate mucosal damage.
Gluten consists of gliadins and glutenins, the water/salt insoluble fractions of storage proteins present in cereal grains. A gluten network is created by interaction between the two proteins when flour and water are mixed in the preparation of dough.
Once ingested, gluten goes towards a partial digestion by gastric-pancreatic and brush-border proteolytic enzymes which results in many peptides of different length (few to more than 30 aminoacids) which are resistant to further digestion due to the high content of proline residues as many proteases are unable to cleave peptide bonds located at N— or C-termini of proline (Hausch F., Shan L., Santiago N. A., Gray G. M., Khosla C. “Intestinal digestive resistance of immunodominant gliadin peptides”. Am. J. Physiol. Gastrointest. Liver Physiol., 2002, 283, 996-1003; Shan L., Molberg O., Parrot I., Hausch F., Filiz F., Gray G. M., Sollid L. M., Khosla C. “Structural basis for gluten intolerance in celiac sprue” Science, 2002, 297, 2275-2279). The lack of proline-specific cleaving enzymes is not a specific enzyme deficiency in celiac subjects, as suggested in the past, but is proper of the mammalian digestive apparatus which has not evolved to consume proteins with so high proline content in its diet.
Undigested gluten peptides can pass the epithelial barrier through mechanisms not yet clearly explained, although a zonulin-mediated paracellular passage and a transcellular way, transcytosis and retrotranscytosis, have been recently proposed (Drago S., El Asmar R., Di Pierro M., Grazia Clemente M., Tripathi A., Sapone A. Thakar M., Iacono G., Carroccio A., D'Agate C., Not T., Zampini L., Catassi C., Fasano A. “Gliadin, zonulin and gut permeability: Effects on celiac and non-celiac intestinal mucosa and intestinal cell lines” Scand. J. Gastroenterol., 2006, 41, 408-19; Schumann M., Richter J. F., Wedell I., Moos V., Zimmermarm-Kordmann M., Schneider T., Daum S., Zeitz M., Fromm M., Schulzke J. D. “Mechanisms of epithelial translocation of the alpha(2)-gliadin-33mer in coeliac sprue” Gut, 2008, 57, 747-754; Matysiak-Budnik T., Moura I. C., Arcos-Fajardo M., Lebreton C., Ménard S., Candalh C., Ben-Khalifa K., Dugave C., Tamouza H., van Niel G., Bouhnik Y., Lamarque D., Chaussade S., Malamut G., Cellier C., Cerf-Bensussan N., Monteiro R. C., Heyman M. “Secretory IgA mediates retrotranscytosis of intact gliadin peptides via the transferrin receptor in celiac disease” J. Exp. Med., 2008, 205, 143-154).
Once in the lamina propria (LM), deamidation of glutamine to glutamate residues by tissue transglutaminase (tTG, the autoantigen in CD) reinforces their presentation to DQ2 or DQ8 CD4+ T cells (Molberg O., McAdam S., Lundin K. E. Kristiansen C., Arentz-Hansen H., Kett K., Sollid L. M. “T cells from celiac disease lesions recognize gliadin epitopes deamidated in situ by endogenous tissue transglutaminase” Eur. J. Immunol., 2001, 31, 1317-23) producing a pro-inflammatory response with interferon-gamma (IFN-γ) as main cytokine effector. Several gluten peptides have also been shown to cause mucosal damage independently from a specific recognition by CD4+ T-lymphocytes, but inducing an innate immune response by up-regulating the expression of IL-15, cyclo-oxygenase-2 and the activation markers CD25 and CD83 in LM mononuclear cells: among them, the best characterized are peptides p31-43/49 of the α1-gliadins (ePGQQQPFPPQQPY/PQPQPF) (Ciccocioppo R., Di Sabatino A., Corazza G. R. “The immune recognition of gluten in coeliac disease” Clin. Exp. Immunol., 2005, 140, 408-16).
CD is estimated to affect about 1% of both European and North American population, with a study from Finland showing increasing rates (1:47) in elder people (Vilppula A., Kaukinen K., Luostarinen L., Krekelä I., Patrikainen H., Valve R., Mäki M., Collin P. “Increasing prevalence and high incidence of celiac disease in elderly people: a population-based study” BMC Gastroenterol., 2009, 29, 9-49). However, many studies indicate that CD is diffused all over the world with similar prevalence values (Barada K., Bitar A., Mokadem M. A., Hashash J. G., Green P. “Celiac disease in Middle Eastern and North African countries: a new burden?” World J. Gastroenterol., 2010, 16, 1449-57; Dalgic B., Sari S., Basturk B., Ensari A., Egritas O., Bukulmez A., Bans Z., Turkish Celiac Study Group “Prevalence of celiac disease in healthy Turkish school children” Am. J. Gastroenterol., 2011, 106, 1512-7; Makharia G. K., Verma A. K., Amarchand R., Bhatnagar S., Das P., Goswami A., Bhatia V., Ahuja V., Datta Gupta S., Anand K. “Prevalence of celiac disease in the northern part of India: a community based study” J. Gastroenterol Hepatol., 2011, 26, 894-900; Wang X. Q., Liu W., Xu C. D., Mei H., Gao Y., Peng H. M., Yuan L., Xu J. J. “Celiac disease in children with diarrhea in 4 cities in China” J. Pediatr. Gastroenterol. Nutr., 2011, 53, 368-70). No therapies are available at this time and the only remediation to disease is a strict, lifelong gluten-free diet necessary to prevent not only CD specific mucosal damage and consequent malabsorption-related disorders (like iron-deficient anemia or osteoporosis) but also other autoimmune diseases which have been associated with CD, like type 1 diabetes and autoimmune thyroiditis, or heavier complications like enteropathy-associated T-cell lymphomas.
Total avoidance of gluten (safe gluten intake threshold is generally indicated in 50 mg/day, although 10 mg/day is considered more safe) maintains CD in remission in all but a small percentage of patients (2-5%) which suffer of a non-responsive form. Such a diet is, however, strongly demanding for patients, which are restricted in their common activities and suffer from social isolation. The use of gluten as additive in food processes is widespread and is the main cause of unaware ingestion of gluten, making this diet really difficult to maintain.
For these reasons it would be strongly welcome by CD patients any alternative allowing them to assume in their daily diet at least minimal amounts of gluten.
The use of exogenous proteolytic enzymes for gluten detoxification is one of the most promising strategies for CD treatment. Different ways of application can exploit these enzymes potential: treatment of gluten containing flours, before or during dough fermentation, thus going towards the production of “novel food”, as well as concomitant consumption of gluten and suitable proteolytic enzymes, thus going as “food supplement”, similarly to the use of lactase for lactose intolerance. Necessarily, different enzyme properties are requested to meet the different objectives.
The enzymatic approach for CD treatment is based on the demonstration by Shan et al. (Science, 2002) of microbial enzymes' ability to cleave gluten peptides on specific residues and remove toxic/immunotoxic specific peptide sequences. In particular, they showed that an exogenous prolyl-specific endoprotease derived from Flavobacterium meningosepticum (FM-PEP) resulted helpful in the digestion of gliadin peptides. The addition of a PEP either in vitro in the presence of brush border membrane (BBM) extracts or during in vivo perfusion of rat small intestine caused a rapid degradation of the immunodominant 33-mer peptide (“33-mer”) and a loss of its capacity to stimulate gliadin-specific T-cells (Hausch et al., 2002).
Other enzymes of the same family (EC. 3.4.21.26) from other bacterial strains (i.e. Sphingomonas capsulata and Myxococcus xanthus) have been evaluated for this aim by the same authors (Shan L., Marti T., Sollid L. M., Gray G. M., Khosla C. “Comparative biochemical analysis of three bacterial prolyl endopeptidases: implications for coeliac sprue” Biochem. J., 2004, 383, 311-318), Globally, these studies showed substantial differences among the three enzymes with respect to chain-length and subsite specificity and confirmed the potential of oral enzyme therapy, although raised concerns regarding their possible efficacy in-vivo, due to restrictions on substrates specificity, pH of activity (optimal activity at almost neutral pH instead of acidic pH as needed to act in the stomach), long time necessary to complete the digestion of toxic peptides and resistance to degradation by pepsin. A combination of two enzymes with gastric activity and complementary substrate specificity was then suggested (Gass J., Bethune M. T., Siegel M., Spencer A., Khosla C. “Combination enzyme therapy for gastric digestion of dietary gluten in patients with celiac sprue” Gastroenterology, 2007, 133, 472-480): PEP from S. capsulata associated to EP-B2, the glutamine-specific endoprotease B isoform 2 from Hordeum vulgare, a cysteine-protease derived from germinating barley seeds that is activated at acidic pH and by pepsin (Bethune M. T., Strop P., Tang Y., Sollid L. M., Khosla C. “Heterologous expression, purification, refolding, and structural-functional characterization of EP-B2, a self-activating barley cysteine endoprotease” Chem. Biol., 2006, 13, 637-47), showed to be a potentially more potent therapeutic tool. Another study reports that a PEP derived from Aspergillus niger, deploying its main activity under acid conditions in the stomach, can start to degrade gliadin before it reached the intestinal lumen (Stepniak D., Spaenij-Dekking L., Mitea C., Moester M., de Ru A., Baak-Pablo R. van Veelen P., Edens L., Koning F. “Highly efficient gluten degradation with a newly identified prolyl endoprotease: implications for celiac disease” Am. J. Physiol. Gastrointest. Liver Physiol., 2006, 291, G621-9).
These findings have been dealt by several patent documents. In WO2003/068170 (EP572127), inventors claim that administering an effective dose of glutenase to a celiac or dermatitis herpetiformis patient reduces levels of toxic gluten oligopeptides, thereby attenuating or eliminating the damaging effects of gluten. Further support to this approach is given in WO2005/107786 (EP1740197), where pharmaceutical formulations of glutenase enzymes for use in the treatment of celiac or dermatitis herpetiformis patients are disclosed. WO2005/027953 (EP 16663298) describes a treatment with a new prolyl-specific endoprotease from Aspergillus niger (AN-PEP) which resulted helpful in digestion of toxic gluten peptides. WO2005/019251 provides leucine aminopeptidase (LAP) of two different fungal species, Trichophyton rubrum and Aspergillus fumigatus in combination with dipeptidyl peptidase IV (DppIV). These enzymes have been evaluated for cleavage of the 33-mer under neutral pH conditions since the optimal activity of LAPs was estimated around 7.0 with a range of activity between pH 6 and 8, thus precluding or limiting a possible breakdown of gliadin in the gastric fluid.
It has also been shown that A. fumigatus tripeptidyl peptidases can degrade proteins at acidic pH (Reichard U., Lechenne B., Asif A. R., Streit F., Grouzmann E., Jousson O., Monod M. “Sedolisins, a new class of secreted proteases from Aspergillus fumigatus with endoprotease or tripeptidyl-peptidase activity at acidic pHs” Appl. Environ. Microb., 2006, 72, 1739-48). In WO2011/077359 it is provided a kit composed by a prolyl-protease (AfuS28) and at least one tripeptidyl protease belonging to the family of sedolisin to be used as food supplement useful also for CD treatment.
It is clear that the therapeutic value of an enzyme or enzyme composition for CD treatment is related to the enzymes a) being resistant to degradation by other gastrointestinal enzymes, b) being efficient in the environment where 33-mer and the others toxic peptides are produced, and c) exhibiting rapid and high proteolytic activity toward gluten peptides. These enzymes should be active at acidic pH and should be able to access a complex composition of gluten hindered by other components of normal foodstuffs baked or cooked.